In vitro and in vivo investigation of chlorophyll binding sites involved in non‐photochemical quenching in
            
              Chlamydomonas reinhardtii

AbstractUnder high light conditions, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating excess absorbed energy, which is called non-photochemical quenching (NPQ). In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via pH and serves as a quenching site. However, the mechanisms by which LHCSR3 functions have not been determined. Using a combined in vivo and in vitro approach, we identify two parallel yet distinct quenching processes, individually controlled by pH and carotenoid composition, and their likely molecular origin within LHCSR3 from Chlamydomonas reinhardtii. The pH-controlled quenching is removed within a mutant LHCSR3 that lacks the protonable residues responsible for sensing pH. Constitutive quenching in zeaxanthin-enriched systems demonstrates zeaxanthin-controlled quenching, which may be shared with other light-harvesting complexes. We show that both quenching processes prevent the formation of damaging reactive oxygen species, and thus provide distinct timescales and mechanisms of protection in a changing environment.

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From ecophysiology to phenomics: some implications of photoprotection and shade–sun acclimation in situ for dynamics of thylakoids in vitro

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2012.0072 ◽

2012 ◽

Vol 367 (1608) ◽

pp. 3503-3514 ◽

Cited By ~ 26

Author(s):

Shizue Matsubara ◽

Britta Förster ◽

Melinda Waterman ◽

Sharon A. Robinson ◽

Barry J. Pogson ◽

...

Keyword(s):

Membrane Dynamics ◽

Persea Americana ◽

Physiological Data ◽

Photochemical Quenching ◽

Shade Acclimation ◽

Non Photochemical Quenching ◽

Time Frames ◽

Shade Leaves

Half a century of research into the physiology and biochemistry of sun–shade acclimation in diverse plants has provided reality checks for contemporary understanding of thylakoid membrane dynamics. This paper reviews recent insights into photosynthetic efficiency and photoprotection from studies of two xanthophyll cycles in old shade leaves from the inner canopy of the tropical trees Inga sapindoides and Persea americana (avocado). It then presents new physiological data from avocado on the time frames of the slow coordinated photosynthetic development of sink leaves in sunlight and on the slow renovation of photosynthetic properties in old leaves during sun to shade and shade to sun acclimation. In so doing, it grapples with issues in vivo that seem relevant to our increasingly sophisticated understanding of Δ pH-dependent, xanthophyll-pigment-stabilized non-photochemical quenching in the antenna of PSII in thylakoid membranes in vitro .

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Allosteric regulation of the light-harvesting system of photosystem II

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2000.0698 ◽

2000 ◽

Vol 355 (1402) ◽

pp. 1361-1370 ◽

Cited By ~ 133

Author(s):

Peter Horton ◽

Alexander V. Ruban ◽

Mark Wentworth

Keyword(s):

Photosystem Ii ◽

Xanthophyll Cycle ◽

Light Harvesting ◽

Allosteric Regulation ◽

Photochemical Quenching ◽

Protein Subunit ◽

Ps Ii ◽

Non Photochemical Quenching

Non–photochemical quenching of chlorophyll fluorescence (NPQ) is symptomatic of the regulation of energy dissipation by the light–harvesting antenna of photosystem II (PS II). The kinetics of NPQ in both leaves and isolated chloroplasts are determined by the transthylakoid ΔpH and the de–epoxidation state of the xanthophyll cycle. In order to understand the mechanism and regulation of NPQ we have adopted the approaches commonly used in the study of enzyme–catalysed reactions. Steady–state measurements suggest allosteric regulation of NPQ, involving control by the xanthophyll cycle carotenoids of a protonationdependent conformational change that transforms the PS II antenna from an unquenched to a quenched state. The features of this model were confirmed using isolated light–harvesting proteins. Analysis of the rate of induction of quenching both in vitro and in vivo indicated a bimolecular second–order reaction; it is suggested that quenching arises from the reaction between two fluorescent domains, possibly within a single protein subunit. A universal model for this transition is presented based on simple thermodynamic principles governing reaction kinetics.

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Antenna Protein Clustering In Vitro Unveiled by Fluorescence Correlation Spectroscopy

International Journal of Molecular Sciences ◽

10.3390/ijms22062969 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2969

Author(s):

Aurélie Crepin ◽

Edel Cunill-Semanat ◽

Eliška Kuthanová Trsková ◽

Erica Belgio ◽

Radek Kaňa

Keyword(s):

Fluorescence Correlation Spectroscopy ◽

Higher Plants ◽

Fluorescence Emission ◽

Correlation Spectroscopy ◽

Photochemical Quenching ◽

High Ph ◽

Fluorescence Correlation ◽

Non Photochemical Quenching

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

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Chlamydomonas reinhardtii LHCSR1 and LHCSR3 proteins involved in photoprotective non-photochemical quenching have different quenching efficiency and different carotenoid affinity

Scientific Reports ◽

10.1038/s41598-020-78985-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Federico Perozeni ◽

Giorgia Beghini ◽

Stefano Cazzaniga ◽

Matteo Ballottari

Keyword(s):

Chlamydomonas Reinhardtii ◽

Functional Properties ◽

Carbon Fixation ◽

Thermal Dissipation ◽

Photochemical Quenching ◽

High Sequence Identity ◽

Pigment Analysis ◽

Quenching Efficiency ◽

Non Photochemical Quenching

AbstractMicroalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, their limited biomass productivity represents a bottleneck that needs to be overcome to meet the applicative potential of these organisms. One of the domestication targets for improving their productivity is the proper balance between photoprotection and light conversion for carbon fixation. In the model organism for green algae, Chlamydomonas reinhardtii, a photoprotective mechanism inducing thermal dissipation of absorbed light energy, called Non-photochemical quenching (NPQ), is activated even at relatively low irradiances, resulting in reduced photosynthetic efficiency. Two pigment binding proteins, LHCSR1 and LHCSR3, were previously reported as the main actors during NPQ induction in C. reinhardtii. While previous work characterized in detail the functional properties of LHCSR3, few information is available for the LHCSR1 subunit. Here, we investigated in vitro the functional properties of LHCSR1 and LHCSR3 subunits: despite high sequence identity, the latter resulted as a stronger quencher compared to the former, explaining its predominant role observed in vivo. Pigment analysis, deconvolution of absorption spectra and structural models of LHCSR1 and LHCR3 suggest that different quenching efficiency is related to a different occupancy of L2 carotenoid binding site.

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Structure of SOQ1 lumenal domains identifies potential disulfide exchange for negative regulation of photoprotection, qH

10.1101/2021.03.16.435614 ◽

2021 ◽

Author(s):

Guimei Yu ◽

Xiaowei Pan ◽

Jingfang Hao ◽

Lifang Shi ◽

Yong Zhang ◽

...

Keyword(s):

Biochemical Characterization ◽

Photochemical Quenching ◽

Interface Residue ◽

Domain Interaction ◽

Disulfide Exchange ◽

Redox Active ◽

Non Photochemical Quenching

Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained component of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP. SOQ1 suppresses qH with its lumen-located C-terminal Trx-like and NHL domains. Here we report crystal structures and biochemical characterization of SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are stably associated, with the potential redox-active motif located at their interface. Residue E859 essential for SOQ1 function is pivotal for mediating the inter-domain interaction. Moreover, the C-terminal region of SOQ1 forms an independent -stranded domain, which possibly interacts with the Trx-like domain through disulfide exchange. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighboring domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

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Regulation of plant light harvesting by thermal dissipation of excess energy

Biochemical Society Transactions ◽

10.1042/bst0380651 ◽

2010 ◽

Vol 38 (2) ◽

pp. 651-660 ◽

Cited By ~ 93

Author(s):

Silvia de Bianchi ◽

Matteo Ballottari ◽

Luca Dall’Osto ◽

Roberto Bassi

Keyword(s):

Higher Plants ◽

Current Knowledge ◽

Protein Complexes ◽

Thermal Dissipation ◽

Photochemical Quenching ◽

Knockout Mutants ◽

Pigment Protein Complexes ◽

Non Photochemical Quenching

Elucidating the molecular details of qE (energy quenching) induction in higher plants has proven to be a major challenge. Identification of qE mutants has provided initial information on functional elements involved in the qE mechanism; furthermore, investigations on isolated pigment–protein complexes and analysis in vivo and in vitro by sophisticated spectroscopic methods have been used for the elucidation of mechanisms involved. The aim of the present review is to summarize the current knowledge of the phenotype of npq (non-photochemical quenching)-knockout mutants, the role of gene products involved in the qE process and compare the molecular models proposed for this process.

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The Role of Acidic Residues in the C Terminal Tail of the LHCSR3 Protein of Chlamydomonas reinhardtii in Non-Photochemical Quenching

The Journal of Physical Chemistry Letters ◽

10.1021/acs.jpclett.1c01382 ◽

2021 ◽

pp. 6895-6900

Author(s):

Franco V. A. Camargo ◽

Federico Perozeni ◽

Gabriel de la Cruz Valbuena ◽

Luca Zuliani ◽

Samim Sardar ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Photochemical Quenching ◽

Acidic Residues ◽

Non Photochemical Quenching

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Non-photochemical quenching of chlorophyll a fluorescence: early history and characterization of two xanthophyll-cycle mutants of Chlamydomonas reinhardtii

Functional Plant Biology ◽

10.1071/fp02061 ◽

2002 ◽

Vol 29 (10) ◽

pp. 1141 ◽

Cited By ~ 28

Author(s):

Govindjee ◽

Manfredo J. Seufferheld

Keyword(s):

Chlamydomonas Reinhardtii ◽

Oxygen Evolution ◽

Xanthophyll Cycle ◽

Photochemical Quenching ◽

Chl A ◽

Fluorescence Transient ◽

Chl A Fluorescence ◽

Non Photochemical Quenching

This paper deals first with the early, although incomplete, history of photoinhibition, of 'non-QA-related chlorophyll (Chl) a fluorescence changes', and the xanthophyll cycle that preceded the discovery of the correlation between non-photochemical quenching of Chl a fluorescence (NPQ) and conversion of violaxanthin to zeaxanthin. It includes the crucial observation that the fluorescence intensity quenching, when plants are exposed to excess light, is indeed due to a change in the quantum yield of fluorescence. The history ends with a novel turn in the direction of research — isolation and characterization of NPQ xanthophyll-cycle mutants of Chlamydomonas reinhardtii Dangeard and Arabidopsis thaliana (L.) Heynh., blocked in conversion of violaxanthin to zeaxanthin, and zeaxanthin to violaxanthin, respectively. In the second part of the paper, we extend the characterization of two of these mutants (npq1, which accumulates violaxanthin, and npq2, which accumulates zeaxanthin) through parallel measurements on growth, and several assays of PSII function: oxygen evolution, Chl a fluorescence transient (the Kautsky effect), the two-electron gate function of PSII, the back reactions around PSII, and measurements of NPQ by pulse-amplitude modulation (PAM 2000) fluorimeter. We show that, in the npq2 mutant, Chl a fluorescence is quenched both in the absence and presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). However, no differences are observed in functioning of the electron-acceptor side of PSII — both the two-electron gate and the back reactions are unchanged. In addition, the role of protons in fluorescence quenching during the 'P-to-S' fluorescence transient was confirmed by the effect of nigericin in decreasing this quenching effect. Also, the absence of zeaxanthin in the npq1 mutant leads to reduced oxygen evolution at high light intensity, suggesting another protective role of this carotenoid. The available data not only support the current model of NPQ that includes roles for both pH and the xanthophylls, but also are consistent with additional protective roles of zeaxanthin. However, this paper emphasizes that we still lack sufficient understanding of the different parts of NPQ, and that the precise mechanisms of photoprotection in the alga Chlamydomonas may not be the same as those in higher plants.

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Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2091-2103.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2091-2103

Author(s):

S Türkel ◽

P J Farabaugh

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Binding Sites ◽

Transcriptional Repression ◽

Physiological Control ◽

Reading Frame ◽

Negative Region ◽

Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

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